Fermented probiotic protein plant beverage comprising quinoa and chickpeas

Abstract

The present invention discloses a liquid food composition comprising quinoa and chickpeas, the composition corresponding to a fermented plant beverage with lactic acid bacteria to improve the digestibility of the plant proteins contained therein. The plant beverage provides a probiotic product with a balanced amino acid profile, which is also stable during storage and has desirable organoleptic properties.

Description

[0001] Fermented probiotic protein plant-based drink made from quinoa and chickpeas

[0002] DESCRIPTIVE MEMORANDUM

[0003] TECHNICAL FIELD

[0004] The present invention relates to the food industry, in particular to the field of fermented probiotic plant beverages and desirable organoleptic properties.

[0005] BACKGROUND OF THE INVENTION

[0006] Plant-based beverages (PBBs) have experienced significant growth in recent decades due to the demand for cow's milk alternatives among people with dietary restrictions caused by allergies, lactose intolerance, and those following a vegan or vegetarian lifestyle. PBBs are typically aqueous extracts obtained through the processing of grains, pseudocereals, legumes, and nuts. Furthermore, people with soy or nut allergies, or celiac disease associated with PBBs made from grains like wheat and barley, cannot consume these non-dairy alternatives. However, compared to cow's milk, PBBs often lack nutritional quality due to their low protein content, which is highly dependent on the plant source, incomplete or unbalanced essential amino acid profiles, and low digestibility.Therefore, PBBs cannot be considered as "milk".

[0007] The protein content in plant-based beverages (PBBs) ranges from a minimum of 0.06% in a cereal- or nut-based beverage to a maximum of 4.3% in a legume-based beverage. Different raw materials can be blended to improve the protein content and amino acid profile of PBBs. Some alternatives include blends of coconut and lupin, resulting in a 13% increase in protein content. For example, a 50:50 blend of quinoa and soy resulted in a beverage with approximately 3% protein. In another study, blends of chickpeas and coconut increased the protein content by approximately 1.4% compared to beverages without any added blends. To improve the low digestibility of plant proteins, various technologies have been applied to plant-based matrices in research, including enzyme-assisted extraction and ultra-high-pressure homogenization.Although these techniques have not been standardized, the mechanisms induced by the treatment and the conformational and / or structural changes they cause in the plant proteins of the PBB have been studied in detail.

[0008] State of the Art

[0009] In the current state of the art, there are many developments related to plant-based drinks and / or probiotic drinks.

[0010] Patent application CN116420847A describes a chickpea-based beverage, which does not mention quinoa or any other alternative raw material. The described beverage is produced by fermentation with lactic acid bacteria, Bifidobacterium, Lactobacillus bulgaricus, and Streptococcus thermophilus; Lactobacillus acidophilus is not mentioned among the selected alternatives. While a fermentation temperature of 42°C is selected, it is not the same strain or substrate mixture as in the present invention. Regarding digestibility, it is only mentioned that fermentation of the chickpea ingredients improves their digestibility, but the digestibility of the proteins is not analyzed in detail. Since it contains only chickpeas, the amino acid profile is limited to the 18 amino acids present in chickpea proteins.

[0011] Document CN111713621A describes a beverage obtained from the fermentation of quinoa with the addition of sodium selenite, exhibiting good flavor and an improved nutritional profile. It does not mention the inclusion of chickpeas or other legumes or grains; however, it does indicate the addition of parts of a medicinal plant. The fermentation is carried out with mixtures of lactic acid bacteria, Lactobacillus bulgaricus and Streptococcus thermophilus, although it does not mention Lactobacillus acidophilus. Among the beverage's properties, it is stated that it has a protein content exceeding 3.8% and contains viable bacteria at a concentration of 9.5–9.8 log CFU / mL, but the protein digestibility is not addressed. While it indicates that the fermentation temperature is in the range of 38–43°C, neither the combination of grains nor the bacterial strain matches that described in the present invention.

[0012] In her 2021 thesis, Mireya Mariscal Orduño analyzes the effect of saccharification on raw and extruded chickpea suspensions to evaluate the performance of supernatants subjected to lactic acid fermentations with different strains of Lactobacillus. The document assesses aspects for improvement in fermented plant-based beverages compared to the characteristics of animal milk, although protein digestibility is not evaluated. It only indicates that fermentation and heat treatment improve protein digestibility, based on a literature review. Among the five strains evaluated are Lactobacillus acidophilus LA3 and four strains of Lactiplantibacillus plantarum.

[0013] The scientific publication by Estefanía Valero-Cases et al., Nutrients 2020, 12, 1666, presents the nutritional contribution of fermented non-dairy beverages in terms of their probiotic, prebiotic, and bioactive compound content. It indicates that the probiotic Lactobacillus acidophilus LA-5 maintains its viability under storage conditions similar to those of the product of the present invention. Although the use of Lactobacillus acidophilus for chickpea fermentation is mentioned, with a fermentation temperature of 37°C, fermentation with a pseudocereal is not analyzed. Regarding quinoa, other types of bacteria are described, such as Lactiplantibacillus plantarum, Lacticaseibacillus casei, and Lactobacillus lactis, with fermentation temperatures of 30°C.The digestibility of proteins in both cases is not analyzed, although it is mentioned that the fermentation of plant matrices improves the digestibility of their components and improves the nutritional characteristics.

[0014] In the publication by Mohammad Alrosan et al., Food Chemistry, Vol. 404, Part B, 2023, 134614, the fermentation of protein concentrates using kefir is presented; however, there is no reference to chickpeas or a grain mixture with quinoa. Regarding the components of kefir, only Lactobacillus spp. is mentioned, along with other lactic acid bacteria, acetic acid bacteria, and yeasts; the presence of Lactobacillus acidophilus LA5 is not specifically mentioned. Regarding the quinoa proteins, it is indicated that their quality improved, with protein digestibility increasing from 78.54% to 87.67% after 5 days of fermentation.

[0015] At the commercial level, cow's milk-based products, such as cultured milk and probiotic drinks, have a protein content of between 1.2 and 1.6 g / 100 mL. Plant-based beverages, with the exception of those made with soy, have a low protein content (0.5 to 2.5 g / 100 mL) depending on their plant source, which contributes to a protein deficiency in vegan and / or vegetarian consumers. As for plant-based soft drinks (PBDs) available on the market, made from almonds, rice, oats, peas, or coconut, the protein content ranges from 0.1 to 1.6 g / 100 mL, compared to 0.7 to 3.3 g / 100 mL for soy-based drinks. In the case of plant-based yogurt, the protein content ranges from 2.2 to 6.0 g / 100 mL, although there are versions with a lower content, of 0.3 g / 100 mL.

[0016] Currently, different protein-based biosynthesis (PBB) products have been fermented with Lactobacillus strains, which can grow in different matrices and improve protein hydrolysis in plant systems. However, there is still minimal information on the impact of this bacterial fermentation on the hydrolysis of compounds present in PBB products and its relationship with aqueous extracts composed of mixtures of pseudocereals and legumes. BRIEF DESCRIPTION OF THE FIGURES

[0017] Figure 1. Protein content (expressed as %, g protein / 100 g beverage) of the fermented plant beverage made with different proportions of quinoa (QF) and chickpea (CF). Quinoa flour (90%) mixed with 10% chickpea flour (QF90-CF10), 75% quinoa flour with 25% chickpea flour (QF75-CF25), or 50% quinoa flour with 50% chickpea flour (QF50-CF50).

[0018] Figure 2. Viable lactic acid bacteria (LAB) (expressed in CFU / mL, CFU: Colony Forming Units) in plant-based beverages made with quinoa flour (QF) and chickpea flour (CF) during 10 h of fermentation. Quinoa flour (90%) Quinoa flour (90%) mixed with 10% chickpea flour (QF90-CF10), 75% quinoa flour with 25% chickpea flour (QF75-CF25), or 50% quinoa flour with 50% chickpea flour (QF50-CF50).

[0019] Figure 3. Degree of protein hydrolysis (DH%) in plant-based beverages made with quinoa flour (QF) and chickpea flour (CF) during 10 h of fermentation. Quinoa flour (90%) mixed with 10% chickpea flour (QF90-CF10), 75% quinoa flour with 25% chickpea flour (QF75-CF25), or 50% quinoa flour with 50% chickpea flour (QF50-CF50).

[0020] Figure 4. Viability of lactic acid bacteria in different fermented plant-based beverages made from mixtures of quinoa flour (QF) and chickpea flour (CF) during 50 days of storage at 8°C. Quinoa flour (90%) mixed with 10% chickpea flour (QF90 / CF10), 75% quinoa flour with 25% chickpea flour (QF75 / CF25), or 50% quinoa flour 50% chickpea flour (QF50 / CF50).

[0021] Figure 5. Water retention capacity (WHC%) study of different fermented plant beverages made with mixtures of quinoa flour (QF) and chickpea flour (CF) during 50 days of storage at 8°C. Quinoa flour (90%) mixed with 10% chickpea flour (QF90 / CF10), 75% quinoa flour with 25% chickpea flour (QF75 / CF25), or 50% quinoa flour 50% chickpea flour (QF50 / CF50).

[0022] Figure 6. Viscosity study of different fermented plant beverages made from mixtures of quinoa flour (QF) and chickpea flour (CF) during 50 days of storage at 8°C. Quinoa flour (90%) mixed with 10% chickpea flour (QF90 / CF10), 75% quinoa flour with 25% chickpea flour (QF75 / CF25), or 50% quinoa flour 50% chickpea flour (QF50 / CF50).

[0023] Figure 7. Protein solubility (g soluble protein / g total protein x 100) of quinoa / chickpea-based flours of Chilean origin (Cahuil / Productos Díaz®) using the BCA (bicinconitic acid) method. 100% quinoa flour (100QF); 90% quinoa flour mixed with 10% chickpea flour (90QF-10CF), 75% quinoa flour mixed with 25% chickpea flour (75QF-25CF), 50% quinoa flour mixed with 50% chickpea flour (50QF-50CF) or 100% chickpea flour (100CF).

[0024] Fig. 8. Protein solubility (g soluble protein / g total protein x 100) of commercially sourced quinoa / chickpea-based flours (Extrumol® products, Bolivian quinoa flour with Argentinian chickpea flour) using the BCA (bicinconic acid) method. 100% quinoa flour (100QF-BH); 90% quinoa flour mixed with 10% chickpea flour (90QF-BH-10CF-AH), 75% quinoa flour mixed with 25% chickpea flour (75QF-BH-25CF-AH), 50% quinoa flour mixed with 50% chickpea flour (50QF-BH-50CF-AH), or 100% chickpea flour (100CF-AH).

[0025] Figure 9. Protein solubility (g soluble protein / g total protein x 100) of commercially sourced sonicated quinoa / chickpea-based beverages (Extrumol® products) using the BCA (bicinconitic acid) method. 100% quinoa flour (100QF); 90% quinoa flour mixed with 10% chickpea flour (90QF-10CF), 75% quinoa flour mixed with 25% chickpea flour (75QF-25CF), 50% quinoa flour mixed with 50% chickpea flour (50QF-50CF), or 100% chickpea flour (100CF).

[0026] DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention provides a food composition based on a mixture of legumes and pseudo-cereals, in the form of a plant-based beverage fermented with lactic acid bacteria, which improves the digestibility of the plant proteins it contains. The plant-based beverage (PBB) offers a probiotic product with a balanced amino acid profile, is gluten-free and lactose-free, stable, and achieves desirable organoleptic properties.

[0028] In a preferred embodiment of the invention, the plant-based beverage is made from a mixture of legumes and pseudo-cereals corresponding to quinoa and chickpea flour.

[0029] The scientific basis lies in the fact that the homolactic fermentation process of mixtures of plant sources with lactic acid bacteria allows the release of peptides and amino acids from plant proteins, thus improving the quality of plant protein in terms of greater amino acid bioaccessibility and a higher protein content than that of quinoa alone in the plant-based beverage. This is particularly relevant considering that it is a flour-based product, as protein content would be easier to control and adjust in preparations using protein isolate.

[0030] Quinoa provides protein with a balanced amino acid profile, and chickpeas provide a high level of protein, in addition to their emulsifying properties, which are better than the protein properties of quinoa and are not significantly altered after fermentation.

[0031] The fermentation of the plant-based beverage is carried out using a bacterial strain with probiotic potential for restoring the gut microbiome. Among the suitable homofermentative lactic acid bacteria (LAB) strains that do not produce other alcohols are probiotic strains such as Lactobacillus, Leuconostoc, Pediococcus, and Streptococcus. Homofermentative strains include: Lactobacillus acidophilus, Lactobacillus delbrueckii, Lacticaseibacillus casei, Lactiplantibacillus plantarum, Lactobacillus bulgaricus, and Lactobacillus helveticus. The lactic acid bacterial strain is preferably Lactobacillus acidophilus, and more specifically, Lactobacillus acidophilus LA-5.

[0032] The reasons for using Lactobacillus acidophilus focus on (i) its commercial availability, which offers an advantage over using a native bacterium, which is not domesticated and has unpredictable metabolism. This commercial LAB ensures controlled and reproducible fermentation, allowing for standardized production of the fermented plant-based beverage; (ii) its optimal growth with plant-based energy sources for a suitable homolactic fermentation process; (iii) its proven probiotic properties, ensuring that the plant-based beverage will have this benefit; and (iv) its tolerance to gastrointestinal digestion.

[0033] Product

[0034] The liquid food composition or beverage according to the present invention comprises a pasteurized mixture of 10-90% chickpeas and 10-90% quinoa in a solids-to-water ratio of 1:5 to 1:10 w / v (weight / volume). Preferably, the solids-to-water ratio is 1:7 w / v.

[0035] The mixture is fermented with lactic acid bacteria, where the bacteria are at a concentration of between 1 x 10 5 1 x 10 10 U FC / ml of final product. Preferably, the bacteria are at a concentration of between 1 x 10 7 1 x 10 8 UFC / ml.

[0036] To enhance the organoleptic appeal to the consumer, it is optional to incorporate sweeteners, preferably stevia, or flavorings to improve the acceptability of the product of the invention, preferably coconut, a flavor that has shown good acceptability in fermented cereal-based PBBs. Optionally, preservatives, antioxidants, colorings, or other food-grade additives may also be incorporated.

[0037] Fermentation is carried out with the lactic acid bacteria Lactobacillus acidophilus LA-5. The particle size of the quinoa and chickpea flours and their mixtures is < 500 pm; in a preferred embodiment of the invention, the particle size is < 300 pm, more preferably < 250 pm.

[0038] The mixture of quinoa and chickpea flours in water is mixed before being subjected to fermentation, for example, by homogenization.

[0039] Optionally, it is possible to dilute the mixture once fermentation is complete to achieve the desired concentration of bacteria in the final product.

[0040] Manufacturing process

[0041] Liquid fermentation was carried out using quinoa and chickpea flour blends where the particle size of the quinoa and chickpea flours and their blends was < 500 µm, preferably < 300 µm, and more preferably < 250 µm in water. The quinoa-to-chickpea flour ratio was 10–90% / 10–90% (quinoa flour / chickpea flour), and the water-to-mixture ratio was 1:5 to 1:10 w / v, preferably 1:7. Mixing was performed by stirring or homogenization, followed by heat treatment (pasteurization) under agitation.

[0042] One of the relevant variables is the degree of starch gelatinization, given by the pasteurization conditions of the mixture.

[0043] Lactobacillus acidophilus strains were used in the fermentation, measuring the effect of lactic acid fermentation on the degree of protein hydrolysis in the PBB.

[0044] The process for preparing the liquid food composition preliminarily requires: a. Grinding, fractionation (with a particle size <500 pm, preferably <250 pm), thereby achieving a concentration of dry proteins.

[0045] And then, the process comprises the steps of: b. mixing quinoa flour (QF) and chickpea flour (CF) in a solid-to-water ratio of 1:5 to 1:10 (weight / volume, w / v), where the selected flour has a particle size <500 pm, c. pasteurizing the mixture, at a time / temperature of 60°C / 30 min to 90°C / 5 min, preferably at 75°C for 15 min, d. inoculating the lactic acid bacteria, and e. fermenting the mixture from the previous step at a temperature between 30 and 47°C.

[0046] Fermentation is carried out at a temperature between 30 and 47°C, until a pH below 4.6 is reached. Pasteurization is carried out at a temperature between 60 and 90°C for 5 to 30 minutes.

[0047] The quinoa and chickpea mixture from step a) is mixed before pasteurization or incorporation of the inoculum. Optionally, before fermenting the flour and water mixture, homogenization and sonication are performed (e.g., at 1500 W, 20 Hz, 5 min with 5 s on / off pulses).

[0048] The quinoa is washed before making quinoa flour.

[0049] In a preferred embodiment of the invention, the chickpeas used to make the flour are skinless or skinless or peeled chickpeas are used.

[0050] The strain incorporated into the inoculum corresponds to Lactobacillus acidophilus LA-5.

[0051] Once fermentation is complete, the product is optionally diluted to achieve a bacterial concentration in the final product of between 1 x 10 5 1 x 10 8 UFC / ml.

[0052] Furthermore, by evaluating the impact of the quinoa and chickpea mixture on the protein content of the beverages and the fermentation performance characterized by lactic acid concentration, as well as verifying changes in pH and titratable acidity (TAA), it was confirmed that the quinoa / chickpea mixture was a feasible and advantageous substrate for the growth of Lactobacillus acidophilus. (See results in Table 2 and Figure 2, which demonstrate the bacteria's ability to utilize carbohydrates and increase in cell concentration.) The incorporation of chickpeas affected the metabolic capacity of Lactobacillus acidophilus (the bacteria's ability to better metabolize the carbon and protein sources of chickpeas compared to those of quinoa), resulting in a significant variation in lactic acid production and protein solubility.Furthermore, fermentation increased the % degree of hydrolysis (DH) of the mixed extracts, suggesting an improvement in the plant-based protein properties of the prepared PBBs.

[0053] Furthermore, the addition of chickpea flour improved the total protein content in the PPB. The finding showed that the QF50 / CF50 ratio had higher protein content, protein solubility, and % DH than the other mixtures.

[0054] The sonication process increased the solubility of proteins in all the commercial flour mixtures analyzed (see Fig. 9).

[0055] Advantages

[0056] Being soy-free, it is a plant-based beverage (PBB) that does not contain soy allergens and is reduced in anti-nutrients, which can cause adverse effects after consumption. Some of these compounds can be reduced through processes such as washing, cooking, or other heat treatments, as is the case with trypsin inhibitors, given that they are heat-labile compounds.

[0057] Phytates are heat-resistant antinutrients found in legumes and grains that negatively interfere with the absorption of minerals such as iron and zinc. They can also form macromolecular complexes with starch and proteins, decreasing their bioaccessibility during digestion. Soybeans have the highest phytate content among legumes, and these levels are not reduced by the pasteurization process commonly used for non-fermented soy beverages. Soybeans also exhibit higher concentrations of the antinutrients they share with quinoa and chickpeas. For soybeans and chickpeas, methods such as soaking, cooking, and sprouting can eliminate these antinutrients.

[0058] Furthermore, compared to chickpeas, soybeans have a higher content of a compound called soy agglutinin, which can have adverse effects on intestinal health as it alters the balance of the intestinal microbiota and modifies its function as a protective barrier against infectious agents.

[0059] Legumes contain phytoestrogens, plant-based compounds that share structural similarities with the hormone estradiol, and therefore can produce estrogenic effects in the body. The combination of quinoa and chickpeas has a low phytoestrogen content, ranging from 0.034 to 1.42 mg / 100g, compared to soybeans, which range from 16.1 to 98.8 mg / 100g. Among the classes of phytoestrogens, isoflavones are the most abundant compounds found in plants and also exhibit the greatest estrogenic activity. These include (in order of estrogenic potency): genistein, equol, glycitein, daidzein, formononetin, and biochanin A.

[0060] Saponins are present in a wide variety of plant-based foods. When consumed in high concentrations, saponins can damage the intestinal lining and cause hemolysis of red blood cells, although in appropriate doses, positive effects such as anti-inflammatory and antibacterial properties have been reported.

[0061] In terms of organoleptic properties, quinoa and soy saponins, present in high concentrations, and chickpea tannins are characterized by a bitter taste. While the presence of saponins is currently most commonly associated with quinoa, other plant-based foods, such as soy, also have higher concentrations. Washing quinoa beforehand significantly removes saponins, and fermentation helps to mitigate adverse flavors and / or aromas. EXAMPLES

[0062] Example 1. Preparation of a batch of fermented plant-based beverage (PBB) according to the invention.

[0063] 1.1. Flour preparation

[0064] Quinoa seeds were washed with water 1:5 (weight:volume) to remove soluble antinutrients such as saponins and then dried for 24 h at 40 ± 2°C using an IN55 incubator (Memmert, Schwabach, Germany). Subsequently, the dried quinoa seeds and dehulled chickpea grains were ground using a laboratory-scale mill (Pulverisette 16, Fritsch, Germany) and sieved through a <250 µm sieve.

[0065] The approximate composition of the samples was determined according to the methods described by AOAC as follows: protein content by the Kjeldahl method (%N x 6.25), moisture content by oven drying at 105°C for 24 h, fat content by Soxhlet extraction, and ash content by muffle furnace at 550°C. The nitrogen-free extract (NFE) was determined by weight difference, subtracting the total weight of the other components quantified in the samples. In addition, the starch gelatinization temperature was determined by differential scanning calorimetry (DSC) (DSC 1 STAR System, Mettler-Toledo, Greinfensee, Switzerland). All analyses were performed in triplicate.

[0066] 1.2. Preparation of fermented plant-based drinks.

[0067] Lactobacillus acidophilus LA-5 (CHR. Hansen, Hdrsholm, Denmark) was cultured in Man Rogosa and Sharpe broth (MRS, Condolab, Madrid, Spain) at 38 ± 2°C for 18 h. Quinoa flour (QF) and chickpea flour (CF) were mixed in a solid-to-water ratio of 1:7 (weight / volume, w / v). Three different ratios of QF and CF were used as aqueous extracts: quinoa flour (90%) was mixed with 10% chickpea flour (QF90 / CF10), 75% quinoa flour with 25% chickpea flour (QF75 / CF25), and 50% quinoa flour with 50% chickpea flour (QF50 / CF50) (% weight / weight, w / w). Aqueous extracts were pasteurized in a WNB 14 water bath (Memmert, Schwabach, Germany) at 75°C for 15 min and homogenized at 14,000 revolutions per minute (rpm) using an OV5 homogenizer (VELP Scientific, Inc., Suffolk, NY, USA). Lactobacillus acidophilus was inoculated into the prepared aqueous extracts at a concentration of 1 x 10⁻⁵. 6CFU / ml (10% volume / volume, v / v). Mixtures were fermented at 38 ± 1°C, 100 rpm, and in an SI500 orbital shaker incubator (Richmond Scientific, Chorley, UK). Samples were taken every 2 h to monitor fermentation kinetics.

[0068] Fermentation is carried out until a pH <4.6 is reached (in accordance with FDA regulations). 1.3. Total protein content and protein solubility

[0069] The protein content of the fermented and unfermented samples is shown in Table 2. In QF90 / CF10, QF75 / CF25, and QF50 / CF50, the total protein content was approximately 1.7%, 2.0%, and 2.2% in the unfermented samples, respectively. At the end of fermentation, the protein content was 1.6%, 1.9%, and 2.3% for QF90 / CF10, QF75 / CF25, and QF50 / CF50, indicating that the fermentation process with Lactobacillus acidophilus did not have a significant impact (p<0.05) on the total protein content of the prepared PBBs. A high proportion of chickpea flour (CF) in the PBBs significantly increased the total protein content. Meanwhile, a higher proportion of quinoa flour (CF) decreased the protein content of the PBBs.

[0070] Following fermentation with lactic acid generation, a significant increase in soluble proteins was observed for the different PBBs prepared (Table 2). At the beginning of fermentation, soluble proteins were lower in the unfermented samples compared to the fermented samples after 10 h (p < 0.05).

[0071] Table 2. Glucose / fructose, lactic acid concentrations, Brix degrees, total protein content and protein solubility of different QF and CF ratios in PBBs. Values ​​are presented as the mean ± standard deviation of three replicates. There were significant differences in the mean values ​​of the same measurement between different prepared PBBs. Lowercase letters indicate significant differences (p < 0.05) within rows.

[0072] 1.4. Degree of protein hydrolysis (%DH)

[0073] Figure 3 shows the % DH in all QF and CF mixtures using Lactobacillus acidophilus LA-5. The % DH increased (p < 0.05) after 6 h of fermentation in all combinations. The % DH was consistent with the lag phases of bacterial growth, decreasing pH, and increasing TTA (titratable acidity), and increased significantly at the end of fermentation, reaching values ​​of approximately 18.1%, 28.8%, and 35.9% in QF90 / CF10, QF75 / CF25, and QF50 / CF50, respectively, after 10 h of fermentation. A higher proportion of QF in the PBB resulted in a lower % DH. Meanwhile, a higher proportion of CF increased the % DH (p < 0.05). The use of Lactobacillus acidophilus LA-5 allowed the fermentation to be completed (p<4.3) in a time of 10 h and improved the degree of hydrolysis of the vegetable proteins.

[0074] Example 2: Preliminary study of protein content and profile of essential and non-essential amino acids

[0075] Protein content

[0076] 2.1 Flours, grains, or seeds: According to bibliographic background, it is possible to affirm that the protein content of plant sources (grains and seeds) is dependent on factors such as variety, ecotype, production area and season of the year.

[0077] Although the protein content of soy exceeds the values ​​reported for chickpeas, soy has a disadvantage in terms of the allergenic capacity of its proteins.

[0078] To improve the bioaccessibility and subsequent bioavailability of protein in plant sources, various processing technologies are used, such as pasteurization, hot water soaking, and high-pressure homogenization. Most of these are thermal processes that can cause denaturation of the proteins present in these plant sources, which is a significant drawback to consider.

[0079] Preliminary study (invention)

[0080] In the case of the present invention, it was determined that there is variability in protein content between the harvest years (2022-2023), being 10.3% and 15.5%, respectively. It should be noted that both batches of quinoa analyzed came from the same area and supplier (quinoa from the San José de Cáhuil Farm), O'Higgins Region, Chile, at coordinates (34°28'44" S 72°00'14" W).

[0081] For chickpeas, the protein content of the flour made from this legume was 16.2%. The peeled chickpeas were obtained from Sociedad Industrial y Comercial Díaz y Cía. Ltd., Santiago, Chile. In this case, no significant differences (p < 0.05) were found between the years (2022-2023).

[0082] In parallel, a comparative analysis of the origin of the plant source (Chile vs. Argentina) was conducted, given the high presence of Argentine chickpeas on supermarket shelves and the fact that Argentina supplements chickpea imports. The results showed that chickpeas from Argentina have a higher protein content (~31.4%) compared to chickpeas of Chilean origin (~26.0%). Both protein contents for chickpeas fall within previously reported ranges.

[0083] 2.2 Plant-based beverages (PBB): According to the bibliographic background available to date, the protein content of PBBs is dependent on both the plant source used and the solid:water ratio that is prepared.

[0084] Quinoa-based PBBs report a protein content of ~0.80% using a 2:30 ratio up to ~1.20% using a 1:7 ratio.

[0085] The plant protein (PV) content in dairy products can be improved by adding protein isolates (e.g., soy protein isolate), germination, and even by using mixtures of different plant sources. However, even if protein content competitive with that of cow's milk (>2.5%) is achieved, the functional properties and the percentage of secondary structure in the PV remain different, negatively impacting protein digestibility (<72%).

[0086] Preliminary study (invention)

[0087] The PBBs of the present invention were made using a ratio of 1:7.

[0088] The PV content present in the quinoa-based PBBs was ~1.47%, while in the chickpea-based PBBs it was ~2.1%; values ​​that are within the range reported in the literature.

[0089] Amino acid profile [essential (AAEs) and non-essential (AANEs)]

[0090] According to literature reviews, quinoa proteins have a balanced amino acid profile with a composition rich in arginine, histidine, and lysine, similar to the protein balance recommended by the Food and Agriculture Organization of the United Nations (FAO). Chickpea proteins, on the other hand, have a significant content of essential amino acids (EAAs) but are deficient in sulfur-containing EAAs (such as methionine and cysteine).

[0091] Soy proteins also have a balanced essential amino acid profile; however, they contain limiting amino acids, specifically sulfur-containing amino acids (methionine and cysteine). Furthermore, some of PBB's target audience currently rejects soy proteins due to allergies to beta-conglycinin and heat-stable glycine. Additionally, other undesirable organoleptic factors, such as off-flavors (e.g., bean / kibble taste, bitterness, and acidity), contribute to the rejection of this plant-based food source.

[0092] To supplement limiting amino acids and increase free amino acid content, the most studied and applied processes in PBB formulation are high hydrostatic pressure, pulsed electric field, and enrichment with concentrated / isolated PVs. These processes can be costly and impact the final PBB formulation.

[0093] Characterization according to the present invention

[0094] Supplementary table. Amino acid content (mg / 100 g of protein) of PBB with different proportions of quinoa flour (QF) and chickpea flour (CF). Example 3: Protein digestibility - in-vitro analysis

[0095] It is important to mention that, according to the existing literature to date, the differences in protein digestibility in PBBs are due to the plant source used for formulation, processing, and also the methodology used for the study of PBB digestibility.

[0096] Regarding the improvement of the digestibility of plant proteins, the reported processes focus on treatments with atmospheric plasma, soaking in water, bleaching, and filtration.

[0097] Even so, the bioaccessibility of plant proteins is poor, as their secondary structure is characterized by a high content of beta sheets (25-60%) and a low content of alpha helices (15-30%). This is relevant because the hydrophobic nature of the beta sheets present in plant proteins facilitates protein aggregation, with interactions that are more difficult to destabilize, resulting in lower digestibility (<64%).

[0098] Example 4: Degree of protein hydrolysis

[0099] The degree of hydrolysis of vegetable oils, according to current literature, depends on the methodology used for their improvement. These methodologies include the use of exogenous enzymes, ultrasonication, pasteurization, soaking, and blanching, among others.

[0100] This variable is generally measured in protein isolates from plant sources, not in the plant source itself. Only Liu et al. (2023) reported the degree of hydrolysis of a chickpea-based beverage, which yielded a result of 2%. In most studies, the degree of hydrolysis is quantified using an amino acid analyzer, but this is a subject of scientific debate because the degree of hydrolysis is defined as "the percentage of peptide bonds broken in relation to the original protein," while the amino acid analyzer quantifies the amount of free amino acids in the sample.

[0101] Preliminary study (invention)

[0102] The degree of hydrolysis at the end of fermentation using Lactobacillus acidophilus LA-5 as a starter inoculum was 18.1%, 28.8%, and 35.9% in PBB with QF9O / CF1O, QF75 / CF25, and QF5O / CF5O.

[0103] • (QF9O / CF1O): 90% quinoa flour mixed with 10% chickpea flour.

[0104] • (QF75 / CF25): 75% quinoa flour mixed with 25% chickpea flour.

[0105] (QF50 / CF50): 50% quinoa flour mixed with 50% chickpea flour.

[0106] Example 5: Comparative analysis of beverage properties: protein solubility and viscosity

[0107] 5.1 Protein solubility

[0108] The solubility of PVs is influenced by numerous factors, including the amino acid composition and sequence, molecular structure, pH, ionic forces of the medium, surface charges, temperature, and the extent of protein aggregation, among others.

[0109] It is important to mention that, as described in the state of the art, the protein solubility of PVs in PBBs can be impacted by the prior processing of the raw material and its formulation (soaking, pasteurization, pH).

[0110] Preliminary study (invention)

[0111] The protein solubility in the unfermented PBB was 5.2%, 5.4%, and 10.5% in QF90 / CF10, QF75 / CF25, and QF50 / CF50.

[0112] • (QF90 / CF10): 90% quinoa flour mixed with 10% chickpea flour.

[0113] • (QF75 / CF25): 75% quinoa flour mixed with 25% chickpea flour.

[0114] • (QF50 / CF50): 50% quinoa flour mixed with 50% chickpea flour.

[0115] Whereas, after 10 h of fermentation with Lactobacillus acidophilus LA-5, the protein solubility was 32.1%, 35.1% and 43.6% in QF90 / CF10, QF75 / CF25, and QF50 / CF50. Measured by the Bradford method.

[0116] That is, after fermentation, an increase in protein solubility of 6, 6.5, and 4 times the initial values ​​is achieved, respectively.

[0117] 5.2 Viscosity

[0118] Viscosity depends on the solid:water ratio, the particle size of the plant source, pH, protein concentration in the system, the degree of starch gelatinization, and the measurement temperature.

[0119] The PVs found in PBBs exhibit low emulsifying capabilities and generate systems with low viscosity. This instability can be avoided by adding emulsifying / stabilizing agents, such as gums, modified starch, carrageenan, or alginates. These compounds act primarily by forming a continuous-phase network, immobilizing water molecules and thus preventing aqueous phase separation in the PBBs.

[0120] Preliminary study (invention)

[0121] The range of variation of these viscosity values ​​is influenced by the measurement temperature and the solids:water ratio in the system.

[0122] The PBBs according to the present invention were made using a ratio of 1:7.

[0123] The viscosity in the unfermented BVs was 9.3 Pa.s, 13.11 Pa.s and 9 Pa.s in QF9O / CF1O, QF75 / CF25, and QF5O / CF5O.

[0124] • (QF9O / CF1O): 90% quinoa flour mixed with 10% chickpea flour.

[0125] • (QF75 / CF25): 75% quinoa flour mixed with 25% chickpea flour.

[0126] • (QF50 / CF50): 50% quinoa flour mixed with 50% chickpea flour.

[0127] After 10 h of fermentation with Lactobacillus acidophilus LA-5, the viscosity was 16.5 Pa.s, 14.62 Pa.s and 18.05 Pa.s in QF90 / CF10, QF75 / CF25, and QF50 / CF50. These viscosity values ​​were measured simulating refrigeration temperatures of 8°C.

[0128] That is, after fermentation, an increase in viscosity of 77%, 12% and 101%, respectively, is achieved above the initial values.

[0129] Figure 4 shows the viability of the bacteria, and Figures 5 and 6 show the water retention capacity and viscosity, respectively, during 50 days of storage at 8°C.

[0130] Example 6: Comparative analysis of PBB properties using commercial flours of diverse origin

[0131] The protein content of commercial flours (from Extrumol® products, with quinoa flour of Bolivian origin (QF-BH) and chickpea flour of Argentine origin (CF-AH)) was evaluated to validate the impact of the mixtures with the increase in protein content in PBB.

[0132] The solubility of proteins from Chilean flours (Figure 7) and from a mixture of Bolivian quinoa flour and Argentinian chickpea flour (Figure 8) was analyzed. Additionally, Figure 9 shows the protein solubility of the sonicated beverages after pasteurization (a process where proteins aggregate and lose solubility). The results showed that sonication increased the solubility of pasteurized proteins in all commercial flour mixtures.

Claims

CLAIMS 1. A liquid food composition, CHARACTERIZED in that it comprises a pasteurized mixture of between 10-90% chickpeas and between 10-90% quinoa in water in a solids-to-water ratio of 1:5 to 1:10 w / v (weight / volume) fermented with lactic acid bacteria, wherein the bacteria are at a concentration of between 1 x 10 5 1 x 10 8 UFC / ml, where chickpeas and quinoa are present as flour with a particle size <500 pm.

2. The composition according to claim 1, CHARACTERIZED in that the lactic acid bacteria corresponds to Lactobacillus acidophilus LA-5.

3. The composition according to claim 1, CHARACTERIZED in that the particle size in the mixture of quinoa and chickpea flours is less than or equal to 500 pm.

1. The composition according to claim 1, CHARACTERIZED in that the mixture of quinoa and chickpea flours in water is mixed by homogenization before carrying out fermentation.

4. The composition according to claim 1, CHARACTERIZED in that the bacteria are at a concentration of between 1 x 10 7 1 x 10 8 UFC / ml.

5. The composition according to claim 1, CHARACTERIZED in that it optionally contains additives of the type sweeteners, flavorings, aromas, preservatives, antioxidants and / or colorings.

6. The composition according to claim 1, CHARACTERIZED in that the solid to water ratio is 1:7 weight / volume.

7. The composition according to claim 3, CHARACTERIZED in that the particle size in the mixture is less than or equal to 250 pm.

8. The composition according to claim 1, CHARACTERIZED in that the chickpeas used are skinless chickpeas.

9. A process for preparing a liquid food composition, CHARACTERIZED in that it comprises the steps of: a. mixing quinoa flour (QF) and chickpea flour (CF) in a solid-to-water ratio of 1:5 to 1:10 (weight / volume, w / v), wherein the selected flour has a particle size <500 µm, b. mixing or homogenizing and pasteurizing the mixture at a time / temperature of 60°C / 30 min to 90°C / 5 min, c. inoculating the lactic acid bacteria, and d. ferment the mixture from the previous stage at a temperature between 30 and 47°C.

10. The process according to claim 9, CHARACTERIZED in that the fermentation is carried out at a temperature between 35 and 45°C.

11. The process according to claim 9, CHARACTERIZED in that the fermentation is carried out until a pH of less than 4.6 is reached.

12. The process according to claim 9, CHARACTERIZED in that the pasteurization is carried out at a temperature between 60 to 90°C for 5 to 30 min.

13. The process according to claim 9, CHARACTERIZED in that the strain incorporated in the inoculum corresponds to Lactobacillus acidophilus LA-5.

14. The process according to claim 9, CHARACTERIZED in that the quinoa and chickpea mixture of step a) is mixed by stirring or homogenized before pasteurizing or incorporating the inoculum and, optionally, homogenization and sonication of the mixture is carried out before incorporating the inoculum.

15. The process according to claim 9, CHARACTERIZED in that the quinoa is washed and dried before making quinoa flour.

16. The process according to claim 9, CHARACTERIZED in that once fermentation is complete, the product is optionally diluted to achieve a bacterial concentration in the final product of between 1 x 10 5 1 x 10 8 UFC / ml.

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